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Research Article *Corresponding author Periyakali Saravana Bhavan, Department of Zoology, Bharathiar University, Coimbatore - 641046, Tamil In vitro and In vivo Nadu, India, Tel: 91-422-2428495/91-9842498138; Email:

Submitted: 26 September 2017 Antibacterial Activity of Marine Accepted: 13 October 2017 Published: 16 October 2017 Alga ornata against ISSN: 2333-7117 Copyright Pseudomonas aeruginosa © 2017 Bhavan et al. OPEN ACCESS in the Freshwater Prawn Keywords • T. ornata • M. rosenbergii Macrobrachium rosenbergii • P. aeruginosa • Survival 1 1 Gopalan Rajkumar , Periyakali Saravana Bhavan *, Veeran • Brown spot Srinivasan2, Annamalai Asaikutti1, and Rajendran Udayasuriyan1 • Hepatopancreas 1Department of Zoology, Bharathiar University, India • Protein 2Department of Science, Bharathidasan University, India

Abstract The in vitro and in vivo antibacterial activity of marine alga, Turbinaria ornata against Pseudomonas aeruginosa on Macrobrachium rosenbergii was studied. The hexanic, acetonic and methanolic extracts of T. ornata were subjected to in vitro study, which showed that the methanolic extract of T. ornata was worked well and produced 24.02 ± 0.47mm zone of inhibition against P. aeruginosa. In the in vivo study, the characteristic appearance of brown/black spots was detected all over the body of M. rosenbergii when the prawns were subjected to immersion in water containing P. aeruginosa (107 cells/mL) and fed with basal diet contained no incorporated T. ornata. Activities of metabolic enzymes (GOT and GPT) and antioxidant enzymes (SOD and catalase), and lipid peroxidation (LPO) were found to be significantly increased in the hepatopancreas of M. rosenbergii due to the infection with P. aeruginosa. These effects were greatly reduced/ neutralized when M. rosenbergii fed with methanolic extract of T. ornata incorporated feed, which in turn ultimately enhanced the survival rate of M. rosenbergii against P. aeruginosa infection. The 2D gel electrophoresis revealed degradation of hepatopancreatic proteins in P. aeruginosa infected M. rosenbergii, whereas the reverse was seen in methanolic extracts of T. ornata incorporated feed fed prawns. Thus, T. ornata possessed the potency of anti P. aeruginosa. One of the active principles of T. ornata, 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-,[R-[R*,R*-(E)]] was found to be bind with the protein, exotoxin-A of P. aeruginosa when molecular docking was performed. Therefore, there is scope for developing aquaculture medicine with T. ornata against Pseudomonas infection.

ABBREVIATIONS The bacterial disease associated with Aeromonas and other PL: Post Larvae; SOD: Superoxide Dismutase; CAT: Catalase; genera of chitinolytic bacteria (Pseudomonas, Vibrio, Beneckea GOT: Glutamic Oxaloacetic Transaminase; GPT: Glutamic Pyruvic and Leucothrix) leaves brown/black spots, necrosis in the Transaminase; LPO: Lipid Peroxidation; 2D gel: 2 Dimensional hepatopancreas and gill disablement [6,7]. A pathogenic Gram Gel Electrophoresis negative bacterium, Pseudomonas aeruginosa can be found in water, soil and sediments [8,9]. Therefore, incidence of P. INTRODUCTION aeruginosa Aquaculture has improved the socio-economic status of prawn M. rosenbergii have been reported [10-13]. Its control is a infections in fish and shellfish including freshwater rural people [1]. The freshwater prawn Macrobrachium challenging task. (Palaemonidae), distributed throughout the tropical and About 6000 of different marine algae have been subtropical zones of the world. Worldwide, 100 species of reported under green, brown and red categories. They are used freshwater prawns have been recorded, among them 40 as food, feed and fertilizer. They have biomedical potentials due species are found in India [2]. Macrobrachium rosenbergii is a potential candidate species for aquaculture, because it provides anti-microbial, anti-viral and anti-tumoral agent [14]. Therefore, nutritious delicacy for human being, and fetched for good price theto presence present ofstudy many aimed bioactive to examine principles the as antibacterial anti-inflammatory, activity with high demand in both domestic and export markets [3]. of the brown alga Turbinaria ornata by their in vitro (by using Disease outbreak due to viral, bacterial and fungal infections hexanic, acetonic and methanolic extracts) and in vivo activity against Pseudomonas aeruginosa induced infection in M. aquaculture, which has become an issue of global concern [4,5]. rosenbergii PL, offering 1.5% methanolic extract of T. ornata threatened the sustainability and profitability of crustacean

Cite this article: Rajkumar G, Bhavan PS, Srinivasan V, Asaikutti A, Udayasuriyan R (2017) In vitro and In vivo Antibacterial Activity of Marine Alga Turbi- naria ornata against Pseudomonas aeruginosa in the Freshwater Prawn Macrobrachium rosenbergii. JSM Biotechnol Bioeng 4(2): 1080. Bhavan et al. (2017) Email:

Central Bringing Excellence in Open Access incorporated diet. In order to reveal this, morphological changes, Experimental feeds activities of antioxidant enzymes [superoxide dismutase (SOD) The following branded feed basal ingredients (BI) were and catalase (CAT)] and metabolic enzymes [glutamic oxaloacetic used to formulate the experimental feed. For protein sources, transaminase (GOT) and glutamic pyruvic transaminase (GPT)], lipid peroxidation (LPO), 2D-gel pattern of hepatopancreas, (25%) were taken. Wheat bran (10%) was used as carbohydrate and molecular docking of the ligand, 2-Hexadecen-1-ol, fish meal (25%), groundnut oilcake (25%) and soybean meal 3,7,11,15-tetramethyl-,[R-[R*,R*-(E)]] (one of the active principle compound of T. ornata) with the protein, exotoxin-A of P. source. For lipid source, Sunflower oil (2%) was taken. Tapioca aeruginosa were studied. oilcake,flour (5%) soy and bean egg meal,albumin wheat (7%) bran were were used thoroughlyas binding agents. mixed. The powdered basal ingredients, such as fish meal, groundnut MATERIALS AND METHODS Then sterilized water was used to prepare the dough, which was Collection and identification of marine alga oil was added with the dough and mixed well. Then, methanolic extractsteam cooked of T. ornata and cooled was separately at room temperature. incorporated Then with Sunflowerthe dough The marine alga Turbinaria ornata was collected from the intertidal region of Mandapam coast (Lat. 9°17’N; Lon. 79°19’E) egg albumin were added and mixed well. Finally, 1% of vitamin of Gulf of Mannar, south-east coast of Tamil Nadu, India. The B-complexat concentration forte withof 1.5% vitamin and mixedC (BECOSULES well. Then® tapioca flour and Ltd., Navi Mumbai, India) and a pinch of salt were also added and thoroughly mixed. Sterilized water was adequatelyCAPSULES, addedPfizer macroalga species was identified based on its morphology by using published by Central Marine Fisheries Research Institute (ICAR), for maintaining the mixer in moist and paste form. This was Kochi,identification India. Finally, manual the of “Economically species was authenticated Important Seaweeds” by Botanical [15] Survey of India (BSI), Coimbatore, India. The pellets were immediately dried in a thermostatic oven at 37- pelletizedº in a manual pelletizer fixed with 3mm diameter mesh. Preparation of extracts of T. ornata 40 C for one hour to quickly reduce the moisture in order to keep them intact, and then shade dried until they reached constant The collected sample was cleaned well with seawater to weight. To maintain its brittleness and prevent fungal attack they remove all the extraneous matter such as epiphytes, sand particles were kept in air tight jars and stored at -20ºC, and used afresh. The and necrotic parts, and brought to the laboratory in plastic proximate composition of organic matters present in the basal bags. The sample was then thoroughly washed with freshwater, diet formulated was determined by adopting the methodology blotted, spread out and shade dried at room temperature for 2 of Castell and Tiews [18] as given in AOAC [19] manual, which

9% total ash, 8.6 % moisture, 32.9% carbohydrate (total nitrogen weeks,The and powdered was ground sample to fine of powder. T. ornata (75g) was separately freecontains extract) 40.5% and crude gross protein,energy, 4281kcal/kg.5.6% crude fat, 3.4% crude fibre, was done with 450ml (1:6 w/v) of hexane, acetone and methanol Experimental animal packed in Whatmann No. 1 filter paper and Soxhlet extraction individually for 9 h each (36 cycle) until a clear colorless solution The post larvae (PL-10) of the freshwater prawn, Macrobrachium rosenbergii were procured from ADAK Hatchery, muslin cloth, concentrated at 40-50°C using rotary vacuum Odayam, Varkala, Thiruvananthapuram, Kerala, India. They evaporatorwas obtained. (ROTAVAP) These extracts attached were withfiltered ultra-cryostat by using double and driedlayer at 40°C under hot air oven [16]. The dark, gummy solid obtained with oxygenated water and acclimatized to ambient laboratory was used for further investigation. conditionswere transported for 2 weeks to in the cement laboratory tank (6 in × 3 polythene × 3 feet) with bags ground filled water (temperature, 27 ± 1.0; pH, 7 ± 0.15; total dissolved solids, Determination of in vitro antibacterial activity 0.9 ± 0.005g L-1; dissolved oxygen, 7.2 ± 0.55mg L-1; BOD 30.0 ± The in-vitro antibacterial activity was studied by agar 1.30mg L-1; COD, 125.0 ± 3.2mg L-1; ammonia, 0.028 ± 0.004mg well diffusion method [17]. To activate the bacterium before L-1). During acclimatization the prawns were fed with boiled egg inoculation, it was cultured on nutrient broth (Himedia, INDIA) and incubated at 37ºC for 24 h. For inoculums preparation and 50% of tank water was routinely changed every day in order to albumin and artificially formulate feed of our own. More than antibacterial assay approximately, 20ml of the autoclaved medium maintain a healthy environment and adequate aeration was also Mueller-Hinton agar (Himedia, INDIA) was dispensed into sterile an environment devoid of accumulated metabolic wastes. The plates and allowed to solidify under aseptic conditions. Then, the unfedprovided feeds, to faeces,ensure moultsufficient and oxygendead prawns supply if toany the were prawns removed and bacterium Pseudomonas aeruginosa was inoculated and spread by siphoning without disturbing the prawns. with a sterile swab on the surface of agar plates and three wells of 5mm diameter were aseptically made on each plate. The crude Feeding trial extracts of were reconstituted in respective solvents at T. ornata Two groups of PL-30 staged prawns (1.45 ± 0.24cm and 0.10 ± 0.03g) were taken in triplicate experimental set-up. Each 50µl of each solvent extracts of T. ornata. The individual solvents group consisted of 30 PL in an aquarium maintained with 30 a concentration of 250mg/mL. The wells were then filled with (hexane, acetone and methanol) and amoxicillin (100mg/ L of ground water. The group-I served as control and fed with ml) were used as negative and positive controls respectively. feed formulated by using BI only. The group-II was fed with º The plates were incubated overnight at 37 C. The diameter of 1.5% of methanolic extract of T. ornata incorporated diet. The the zones of inhibition was measured and compared to that of water medium was renewed every 24 h by siphoning method positive and negative controls. The experiment was conducted without severe disturbance to the PL and aerated adequately. three times. The test prawns were fed with above prepared diets at 10% of

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Central Bringing Excellence in Open Access body weight twice a day (8.00 a.m. and 8.00 p.m.) consecutively hydroxide solution. The color development was read at 505nm for 60 days and assumed that they have reached PL-90. During using a spectrophotometer within 15 min. Sodium pyruvate (170 the feeding trial, the unfed feed, feces and moults if any were U L-1) was used as a calibrator. The activity of GPT was expressed removed on a daily basis while renewing the aquarium water. as Unit L-1. The similar experimental set-up was maintained then and there Activities of enzymatic antioxidants and lipid to study different parameters. peroxidation Pathogen challenge test Tissues of hepatopancreas (100mg each) was homogenized The pathogenic bacterium, P. aeruginosa isolated and (10% w/v) in ice-cold 50mM Tris buffer (pH 7.4), centrifuged at characterized previously in our laboratory [20] from infected 9329g for 20 min at 4ºC and the supernatant was used to assay (brown/ black spotted) M. rosenbergii taken from the wild the activities superoxide dismutase (SOD) and catalase (CAT). (GenBank accession number, KX398049) was used in this study SOD activity was measured by pyrogallol (10mM) for immersion assay. Culture of P. aeruginosa was maintained autoxidation in Tris buffer (50mM, pH 7.0) by adopting the using nutrient agar. A preliminary bioassay was conducted to method of Marklund and Marklund [23]. The reaction was assess the survival capacity of PL in 1.5ml L-1 cultured broth [21]. initiated by the addition of NADH. The mixture was incubated at Actually, PL was immersed for 2, 4, 6 h duration and allowed to 30°C for 90 sec and arrested by the addition of glacial acetic acid. survive in normal water for 10 days (survival rate, SR = No. of The reaction mixture was then shaken with n-butanol and the live prawns/ No. of prawns introduced × 100). This revealed intensity of the chromogen in the butanol layer was measured that no mortality was occurred in any of the immersion duration. 1, 103, 105 7 -1 enzyme was expressed in unit/ mg protein. and 10 cells mL and the mortality was assessed against 2, 4, 6 at 560nm using spectrophotometer. The specific activity of the Therefore, the concentrations were7 magnified-1 to 10 h immersion durations. Finally, 10 cells mL concentration was CAT activity was measured by using H2O2 as the substrate chosen under 2h immersion duration. in phosphate buffer by adopting the method of Sinha [24]. The reaction was initiated by the addition of phosphate buffer (0.01 The above experimental PL group-II that was in feeding trial M, pH 7.1), H O (0.2 M). After 60 sec the reaction was stopped by for 60 days subjected to immersion assay (107 cells mL-1) for 2 h 2 2 the addition of dichromate acetic acid reagent. The absorbance was extended as challenged treatment group for further 21 days, of the chromophore was read at 620nm. The activity of CAT was non-infected freshwater medium was used for renewal every day and they were continuously fed with 1.5% of methanolic protein. extract of T. ornata incorporated diet. The control (group-I) expressed as μM of hydrogen peroxide consumed/ minute/ mg was divided into two sub-groups, one negative control and one Lipid peroxidation (LPO) in tissue homogenate was measured positive control (immersed/ infected control), simultaneous and by estimating the formation of thiobarbituric acid reactive separately maintained for 21 days, the water was renewed and substances (TBARS), malondialdehyde (MDA) by adopting the feeding was allowed as mentioned earlier with BI. Actually, the method of Ohkawa et al. [25]. The absorbance of the supernatant group-I (control PL) was dived into unchallenged and challenged was measured at 535nm against the reagent blank. TBARS controls. These prawns were observed for morphological was expressed as nM of malondialdehyde (MDA)/ mg protein. changes, appearance of brown/black spots. Concentration of soluble proteins was determined by the method of Lowry et al. [26]. Activities of metabolic enzymes Profiles of proteins The metabolic enzymes such as glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase After 21 days immersion assay, the SDS-PAGE analysis was (GPT) were analyzed by the method of Reitman and Frankel [22] done on the prawns of unchallenged control, challenged control, using a med source kit (Medsource Ozone Biomedicals Pvt. Ltd. and challenged treatment groups. The hepatopancreas tissue Haryana, India). Each 100mg of hepatopancreas tissues were sample was homogenized in phosphate buffer (137mM NaCl, homogenized in 0.25M sucrose and centrifuged at 3300g for 20 2.7mM KCl, 10mM Na2HPO4 and 2mM KH2PO4, pH-7.4) under ice min in a high speed cooling centrifuge at 4ºC. The supernatant cooled condition and centrifuged at 1500rpm at 4ºC for 5 min. was used as the enzyme source. The soluble protein content in supernatant was determined by Bradford [27]. Bradford reagent was prepared by dissolving 100mg Coomassie Brilliant Blue G-250 in 50ml of 95% ethanol º C for and 100ml of 85% phosphoric acid. The mixture was then topped GOT analysis, the substrate solution, L-aspartic acid (500μL; andpH, 7.4)allowed was toadded stand to for a 100μL20 min sample at room and temperature. incubated atThen 37 3mL 1 h. Further, 500μL of 2, 4-dinitrophenyl hydrazine was added of freshly prepared 4 N sodium hydroxide solution was added to light.to 1 L SDS-PAGE with deionized was performed water and [28] stirred on vertical overnight. slab The gel withfinal the above solutions. The color development was read at 505nm 4%mixture stacking was filteredand 10% and separating stored in gels.room Marker temperature consisting away of from six using spectrophotometer within 15 min. Sodium pyruvate (160 different molecular weight proteins (Medox-Biotech Pvt. Ltd., U/L-1) was used as a calibrator. The activity of GOT was expressed -1 as Unit L . albumin), 45kDa (ovalbumin), 29kDa (carbonic anhydrase), GPT analysis, buffered L-alanine, 2-oxoglutarate substrate 20kDaIndia), (soybean such as116kDa trypsin (β-galactosidase), inhibitor) and 14kDa 66kDa (lysozyme) (bovine serum were also ran simultaneously. The polypeptides banding patterns 37º between control and test prawns were compared by using (500μL;was added pH, and 7.4) allowed was added to stand to a at100μL room sample temperature and incubated for 30 min at information on apparent molecular masses of bands and their followedC for 20 by min. the additionWith this, of 500μL 3mL ofof freshly2, 4-dinitrophenyl prepared 4 hydrazineN sodium intensity.

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Central Bringing Excellence in Open Access 2D Gel electrophoresis (pH dependent and anionic and subsequent post hoc multiple comparison, Duncan’s multiple exchange) treatments at P<0.05. After 21 days immersion assay, 2D Gel analysis was done range test (DMRT) to compare the significant differences among on M. rosenbergii of negative control, positive control, and RESULTS challenged prawns fed with 1.5% of methanolic extracts of T. In-vitro antibacterial activity of T. ornata ornata incorporated feed. The hepatopancreas was aseptically removed and extracted with 1% triton X-100 buffer [1% triton Hexanic, acetonic and methanolic extracts of T. ornata X-100, 20mM Tris-HCl (pH -8.0), 150mM NaCl and 10µl protease showed antibacterial activity against P. aeruginosa (Figure 1; inhibitor cocktail]. The soluble protein concentration in the Table 1). This was assessed by comparing the zone of inhibition of standard antibiotic, amoxicillin. The inhibition zone of 19mm, 23mm and 24mm was observed for hexanic, acetonic Rehydration of the dry strips was done in rehydration hepatopancreas was estimated and quantified [27]. and methanolic extracts, respectively. The positive control (amoxicillin) produced 21-24mm zone of inhibition. The negative solution at 250μl per strip in the presence of 8 M urea, 2% v/v controls (hexane, acetone and methanol) did not show any CHAPS, trace of bromophenol blue, 50mM DTT, 2.5μl IPG buffer inhibition. fromand dry-stripeach hepatopancreas cover liquid. samples The first were dimension loaded onto of 2-DE each wasIPG Survival rate and morphology of P. aeruginosa stripperformed (nonlinear in IPGphor pH 3-10; IEF 13cm system. long). A totalThe focusedof 250μg IPG of strip proteins was challenged prawn incubated for 15 min in an equilibration buffer containing 87% w/v glycerol, 60.06 Mw Urea, 2% w/v SDS, 50mM Tris-HCl (pH The survival rate of the uninfected control was higher when 8.8), 100mM DTT, and trace of bromophenol blue. Before running compared with infected control. When the infection due to P. the second dimension the strip was then further equilibrated aeruginosa was challenged with 1.5% of methanolic extract of for 15 min in a similar buffer, which replaced 100mM DTT with T. ornata incorporated feed, the PL showed higher survival rate 250mM of iodoacetamide. The IPG strip was placed onto the top than that of the uninfected control. Therefore, T. ornata has of 12% SDS-PAGE gel and sealed with hot agarose (1% w/v). enhanced the survival rate in M. rosenbergii under infection by P. After the run (up to 8 hr at 100 V DC under air conditioned room), aeruginosa (Table 2). The positive control showed external sign of infection, such as brown/black spots over the body, ruptured uropods and loss of periopods (Figure 2). These signs were not 2O andthe gelstained was fixedwith Coomassiein a fixative Brilliant solution Blue containing G-250 overnight, 500ml ethanol then seen in the uninfected control, nor in the prawns fed with 1.5% of de-stainedand 120ml with acetic solution acid, 500μlcontaining formaldehyde methanol 35%,(43%), 380ml acetic dH acid methanolic extract of T. ornata incorporated feed. (7ml) and double distilled water (50ml) until a clear background Activities of metabolic enzymes, antioxidant and lipid was achieved. All the images were collected on Bio-Red images peroxidation (Bio-Red Laboratories, Inc.). The activities of GOT, GPT, SOD, CAT and LPO were Molecular docking analysis P. aeruginosa infected control (P<0.05) when compared to uninfected control and the prawns fed with significantly1.5% of methanolic increased extracts in of T. ornata incorporated feed andIntermediary grid box creation steps, were such done as PDBQT by using files graphical for protein user (bacterial interface (Table 3), indicating that this marine algae have the capacity to programtoxin) and AutoDock ligands (bioactive Tools (ADT). compound ADT assigned identified) polar preparation hydrogens, neutralize the effect of the pathogenic bacterium, P. aeruginosa. united atom Kollman charges, solvation parameters and Profiles of proteins fragmental volumes to the protein. AutoDock saved the prepared Polypeptide bands between 139-12kDa were resolved in the the grid map using a grid box. The grid size was set to 60 × 60 × hepatopancreas of M. rosenbergii PL (Figure 3). In the uninfected 60file xyzin PDBQT points format.with grid Auto spacing Grid wasof 0.375 used Åfor and the grid preparation center was of control, 14 out of 18 protein bands, were prominently stained designated at dimensions (x, -1.095; y, -1.554 and z, 3.894). A (139, 116, 59, 50, 47, 45, 39, 36, 29, 20, 17, 16, 14 and 12kDa). scoring grid is calculated from the ligand structure to minimize The infected control presented, weaker stain intensity and 139, the computation time. 116 47, 20, 16 and 12kDa protein bands were less visible. P. aeruginosa infection challenged with 1.5% methanolic extract of AutoDock was employed for docking using protein and ligand T. ornata incorporated feeds fed prawns showed several protein bands with high, the stain intensity, such as 139, 116, 66, 61, 59, 56, 50, 47, 45, 32, 29, 20, 19, 17, 16, 14 and 12kDa. [29,30].information During along the with docking grid box procedure, properties both in the the configuration protein and ligandsfile. AutoDock are considered employs as iterated rigid. The local results search less global than optimizer 1.0 Å in 2D Gel electrophoresis positional root-mean square deviation (RMSD) was clustered The hepatopancreas of all three categories of prawns showed, together and represented by the result with the most favorable 24 marked protein spots (Figure 4). All the marked spots were free energy of binding. The pose with lowest energy of binding prominent and clear in the uninfected control (Figure 4a), whereas in the P. aeruginosa infected control, all these protein structure for further analysis. or binding affinity was extracted and aligned with receptor spots were correspondingly dull and dispersed (Figure 4b). In STATISTICAL ANALYSIS T. ornata incorporated feeds fed prawns, all these protein spots were found to be very prominent and clear, similarly to the The data were expressed as mean ± SD (n=3), and analyzed by uninfected control (Figure 4c). one-way analysis of variance (ANOVA) using SPSS (version-20),

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Figure 1 In-vitro antibacterial activity of T. ornata against P. aeruginos: a) Hexanic extract; b) Acetonic extract; c) Methanolic extract.

Table 1: In-vitro antibacterial activity of T. ornata extracts against P. aeruginosa. Extracts concentrations Zone of inhibition (mm) Positive control, Amoxicillin 23.38 ± 1.04 (5mg or 50µl/ well = 100mg/ml Negative controls (Hexane, acetone and methanol: 50µl/ -- well) Hexanic extract 19.01 ± 0.46 (12.5mg or 50µl/ well = 250mg/ml) Acetonic extract 22.52 ± 0.58 (12.5mg or 50µl/ well = 250mg/ml) Methanolic extract 24.02 ± 0.47 (12.5mg or 50µl/ well = 250mg/ml) Each value represents mean ± SD; n=3 (‘n’ for positive control =18). Mean values within the same column sharing the different alphabetical P < 0.05 (one way ANOVA and subsequent post hoc multiple comparison with DMRT). superscripts are statistically significant at Molecular docking

T. ornata were recorded: 4,8,12,16-Octadecatetraen-1-ol, 4,9,13,17-tetramethyl;In our previous study, Neophytadiene; five bioactive 17 compounds Pentatriacontene; from 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-,[R-[R*,R*-(E)]]; and Figure 2 Appearance black/brown spots on M. rosenbergii after Squalene [16]. All of these compounds were taken as ligands challenging study: a) Negative control, looked normal in color; b) and subjected to molecular docking with the protein, exotoxin-A and c) Dorsal and lateral views of positive control, infected with P. from P. aeruginosa aeruginosa, which exhibited brown/black spots over the body; d) Absence of brown/black spots on the body of M. rosenbergii fed with was found to be bind with only one bio-active compound, 1.5% methanolic extracts of T. ornata, which showed resistance to P. . Among the five compounds, *the* exotoxin-A 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-,[R-[R ,R -(E)]]. The aeruginosa infection . gliding score, docking score and length of the H bond were shown

3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]] was found to be bind with Durio zibethinus supplemented diet showed higher resistance activein tables site 4of and GLY 5,441 and (amino figure acid 5. Theof protein, ligand, exotoxin-A) 2-Hexadecen-1-ol, and the against viral and bacterial pathogens. Holdt and Kraan [36] have bond length was 2.182 for hydrogen to oxygen from protein and reported that polysaccharides (alginate, fucoidan and laminarin) 2.147 for sites of ligand from oxygen to hydrogen, respectively. present in algal extracts can control viral and bacterial infections. In the present study, methanolic extract of DISCUSSION T. ornata incorporated diet improved survival performance in M. In the present study, methanolic extracts of T. ornata rosenbergii PL. Immanuel, et al. [37], demonstrated that herbal showed anti-bacterial activity against P. aeruginosa. Bioactive and algal extracts may be effectively used as a dietary source to components present in seaweeds inhibit the growth of some enhance the disease resistance as well as to increase survival and gram-positive and gram-negative bacteria [31]. Marine algal production of Penaeus indicus in aquaculture systems. Manilal extracts have been used as curative and preventive agents et al. [38], contemplated the effect of Acrosiphonia orientalis incorporated feed on the survival rate of P. monodon against hypertension, tumor and diarrhoea [31-34]. Pholdaeng and Vibrio harveyi and Vibrio alginolyticus infections. Similarly, Ulva Pongsamartagainst helminths, [35] reported bacteria, that virus, Penaeus fungi, monodon inflammation, pre-fed cough, with diet at a dose of 1000mg/kg has enhanced the survival rate of P.

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Table 2: Survival rate of M. rosenbergii PL against P. aeruginosa infection treated with methanolic extracts of T. ornata incorporated feed. Time Negative control Positive control T. ornata (Days) (uninfected) (infected) (1.5%) 0 100.00 ± 0.00 a 100.00 ± 0.00 a 100.00 ± 0.00 a 7 100.00 ± 0.00 a 97.77 ± 1.92 a 100.00 ± 0.00 a 14 95.55 ± 1.92 abc 75.55 ± 1.92 d 96.66 ± 3.33 ab 21 81.11 ± 1.92 c 61.11 ± 1.92 d 92.22 ± 1.92 b Each value represents mean ± SD; n=3; Mean values within the same row

P < 0.05 (one way ANOVA and subsequent post hoc multiple comparison withsharing DMRT). different alphabetical superscripts are statistically significant at

Table 3: Activities of antioxidant and metabolic enzymes, and lipid peroxidation in the hepatopancreas of M. rosenbergii PL on 21st day Figure 3 SDS-PAGE (12%) pattern of hepatopancreatic proteins of M. after challenged with P. aeruginosa infection treated with methanolic rosenbergii: M - Marker; Lane-1 - Negative control (uninfected); Lane- extract of T. ornata incorporated feed. 2 - Positive control (infected by ); Lane-3 - P. aeruginosa P. aeruginosa Negative Positive infection challenged with methanolic extract of (1.5%) T. ornata T. ornata Parameters control control incorporated feed. (1.5%) (uninfected) (infected) SOD (µmol /min/mg 14.88 ± 12.56 ± 1.24b 26.76 ± 2.62a protein) 1.93b 24.28 ± CAT (Unit/mg protein) 22.56 ± 2.47b 39.28 ± 3.81a 2.74b LPO (nmol MDA/mg 2.34 ± 1.70 ± 0.08b 8.65 ± 0.31a protein) 0.28b 14.23 ± GOT (Unit/L) 12.17 ± 1.48b 22.83 ± 1.74a 1.58b 14.46 ± GPT (Unit/L) 12.79 ± 1.38b 25.43 ± 2.19a 1.52b Each value represents mean ± SD; n=3; Mean values within the same row sharing different alphabetical superscripts are statistically P < 0.05 (one way ANOVA and subsequent post hoc multiple comparison with DMRT). SOD:significant Superoxide at Dismutase; CAT: Catalase; LPO: Lipid Peroxidation; MDA: Malondialdehyde; GOT: Glutamic Oxaloacetic Transaminase; GPT: Glutamic Pyruvic Transaminase.

monodon against Vibrio infection [39]. Bhavan and Geraldine [40] reported that hepatopancreas is a sensitive organ predisposed to injury by water born diseases 2D gel pattern of hepatopancreatic protein of M. rosenbergii Figure 4 and pollutants. Brown and black spot disease caused by P. (I-dimension: pH dependent; II-dimension: anionic exchange): a) Negative control (uninfected); b) Positive control (infected by P. aeruginosa infection led to pale hepatopancreas, which affect aeruginosa); c) P. aeruginosa infection challenged with methanolic the biosynthesis of proteins, and could have resulted in loss of extract of T. ornata (1.5%) incorporated feed. cellular ions and proteins. Therefore, breakdown/degradation of proteins may have occurred as a physiological stress response in prawns infected with P. aeruginosa. The breakdown of protein might have overcomed its synthesis. Thus, the stain intensity of various polypeptide bands of the hepatopancreas of M. rosenbergii infected with P. aeruginosa was considerably reduced. Protein degradation may occur due to excessive proteolysis to overcome the metabolic stress, as deposited protein in the cytoplasm can easily be used to replace the loss of proteins that occur during physiological stress [41,42]. In this study, the expression of proteins and their alterations Figure 5 3D docking structure of 2-Hexadecen-1-ol, were observed in 24 protein spots in the hepatopancreas of M. 3,7,11,15-tetramethyl-,[R-[R,R-(E)]] of T. ornata with exotoxin-A of rosenbergii infected with P. aerugionsa. Somboonwiwat, et al. P. aeruginosa: Benzine ring, appeared with green color, is the target [43], reported that a total of 27 different protein spots were protein, exotoxin-A of P. aeruginosa. expressed in hemolymph of P. monodon infected with Vibrio

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Table 4: Details of glide score, docking score and hydrogen bond length for ligand and exotoxin-A protein. Glide H Potential Name of compound Dock score Binding energy score Bond energy 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-,[R- -3.0 -3.0 -4.11 125.432 -57.387 [R*,R*-(E)]]

Table 5: Complex of bond length of amino acids for protein and ligand. Interacting Name of compound AA from protein AA from ligand Bond Length residue Hydrogen Oxygen GLY 441 2.182 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-,[R-[R*,R*-(E)]] Oxygen Hydrogen GLY 441 2.147 AA: Amino Acid; GLY: Glycine harveyi. Alinejad, et al. [44], reported that an average 20 out REFERENCES of 300 protein spots in each prawn hemocyte gel underwent 1. . considerable alterations in their expression levels upon infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection 2. http://www.fao.org/fishery/statistics/enMariappan P, Balamurugan P, Balasundaram C. Freshwater prawn, , a promising candidate for rural nutrition. in M. rosenbergii. Macrobrachium nobilii Aquacult Asia. 2003; 8: 13-14.

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Cite this article Rajkumar G, Bhavan PS, Srinivasan V, Asaikutti A, Udayasuriyan R (2017) In vitro and In vivo Antibacterial Activity of Marine Alga Turbinaria ornata against Pseu- domonas aeruginosa in the Freshwater Prawn Macrobrachium rosenbergii. JSM Biotechnol Bioeng 4(2): 1080.

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